Degeneration of retinal neurons is a debilitating condition and a major cause of irriversible blindness worldwide (National Eye Institute, “Vision Research: A National Plan, 1994-1998”). Retinal degeneration is often an endpoint of a variety of ocular and systemic diseases and environmental conditions. Degenerative retinopathies generally affect two neuronal cell populations in the retina: the photoreceptors and ganglion cells. Degeneration of photoreceptors and ganglion cells can arise from neurodegenerative diseases (macular degeneration, glaucoma, retinitis pigmentosa), optic nerve degeneration and optic neuritis, chronic metabolic diseases (proliferative diabetic retinopathy exposure to neurotoxins, ischemia, and physical trauma (Yanoff and Duker, “Ophthalmology,” 8.1.1-8.48.1; National Eye Institute, “Vision Research: A National Plan, 1994-1998”).
Previous work has shown that subconjunctival injections of ATP in rabbits result in increased ATP content in the choroid and retina (Dobromyslov et al., Clinical Conference of May 24, 1966). Dobromyslov et al. also show that subconjunctival injection of ATP ameliorated vision loss in patients suffering from maculodystrophy, pigmental dystrophy, complicated myopia and far advanced glaucoma. (Vestn Oftalmol., Jan.-Feb. 24-6, 1969). Dobromyslov et al. state that a direct makeup of energy reserves occurs in subconjunctival administration of ATP because of fast decomposition of ATP in the retina. The authors postulate that the increase in total retinal ATP levels enhances metabolic support of photoreceptors because rhodopsin hydrolyzes ATP and ATP has a well-known role as an energy source in the cell. Currently subconjunctival injection of ATP is not used for treating retinal dystrophies.
To date the methods for treating retinal degenerations are targeted at activating growth factor receptors, such as glial derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) (Louis, U.S. Pat. No. 5,641,750; LaVail., et al., U.S. Pat. No. 5,667,968). The receptor systems employed by these signaling proteins, GDNF, CNTF, and BDNF, are from a receptor class different from the family of P2Y receptors. Currently, these treatments have not been developed for use in the clinic.
Previous work has demonstrated the presence of P2Y receptors in glial and neuronal cells of the mature nervous system (Abbracchio and Burnstock, Jpn J Pharmacol, 78:113-45, 1998). P2Y receptors belong to a class of G-protein coupled receptors (GPCR) that activate a variety of intracellular signaling pathways. Although features of P2Y receptor signaling in many cell types are well known, the physiological roles of P2Y receptors in the nervous system are not well-characterized. In central, peripheral and sensory nervous systems, P2Y receptor activation profoundly affect glia, a cell type that plays important roles in nervous system development, function, and survival. Previous work has suggested a role for P2Y receptors in neurotransmission, neuronal-to-glial cell-cell signaling, alterations of gene expression, neuritogenesis, and interactions with growth factors in an additive or synergistic manner (Abbracchio and Burnstock, Jpn J Pharmacol, 78:113-45, 1998).
Glial cells in the mature nervous systems provide trophic support to neurons and are therefore a viable cellular target to effect neuronal preservation and survival in a variety of neurodegenerative conditions. Müller cells, astrocytes, and retinal pigment epithelium are three glial cell types in the retina. They form a network of supporting cells around neurons, and this close anatomical proximity is what affords glial cells the capacity to maintain survival and protect neurons. Glial cells provide mechanical support, scavenge debris after neuronal death and injury; provide insulating sheaths for electrical conduction of nerve impulses; buffer extracellular ionic, metabolic, neurotransmitter, and fluid composition; control outgrowth of neurons following injury and death; and provide trophic support for neurons in normal and pathological conditions (Kandel and Schwartz, Principles of Neuroscience, 1979, 2nd Ed., Elsevier, New York, 1985). Under conditions of neurological stress and disease, the extracellular environment surrounding neurons and glia are dramatically perturbed, and glial cells contain a wide array of receptors that can integrate this plurality of external stimuli to mount an appropriate, adaptive response (Kandel and Schwartz, Supra). Adenosine 5′-triphosphate (ATP) is a ubiquitous source of cellular energy and is normally contained inside the cells or can be released extracellularly under regulated conditions (Ralevic and Burnstock, Pharmacol Rev, 50:413-92, 1998). However, under pathological neurological conditions, ATP is believed to be released in an unregulated capacity from damaged cells and can subsequently activate cell surface ATP receptors on glia (Abbracchio and Burnstock, Jpn J Pharmacol, 78:113-45, 1998). The P2Y subtype of nucleotide receptors, which are found on glia, respond to extracellular nucleotides by activating a variety of intracellular signaling pathways and enable the activated glial cell to respond to the underlying neuronal stress and damage (Abbracchio and Burnstock, Jpn J Pharmacol, 78:113-45, 1998).
Uridine 5′-triphosphate (UTP) and ATP have been shown to activate the P2Y subtypes of purinergic receptors in multiple glial cells of the central, peripheral, and sensory nervous systems, including oligodendrocytes, astrocytes, Müller cells, retinal pigment epithelium, and resident macrophages (Abbracchio and Burnstock, Jpn J Pharmacol, 78:113-45, 1998; Kirischuk, et al., J Physiol (Lond),483:41-57, 1995; Liu and Wakakura, Jpn J Ophthalmol, 42:3340, 1998; Lyons, et al., J Neurochem, 63:552-60, 1994; Neary and Zhu, Neuroreport, 5:1617-20, 1994; Peterson, et al., J Neurosci, 17:2324-37, 1997). Mild mechanical forces have been shown to stimulate ATP release from the retina, which could signal through P2Y receptors on retinal neural and glial cells (Jensen, IOVS (suppl.), 40:1237, 1999). Activation of P2Y receptors on glial cells causes concomitant stimulation of phospholipase C and Ca2+ release from intracellular stores and activation of intracellular ras-MAPK (mitogen-activated protein kinase) pathway, both of which are linked to cellular differentiation and survival (Idestrup and Salter, Neuroscience, 86:913-23, 1998; Neary, et al., J Neurosci, 19:4211-20, 1999; Segal and Greenberg, Annu Rev Neurosci, 19:463-89, 1996). Stimulation of DNA synthesis and cell proliferation by purines has been observed in primary astrocytes and astrocytoma cells (Neary, et al, J Neurochem, 63:2021-7, 1994; Neary, et al., J Neurochem, 63:490-4, 1994; Rathbone, et al., In Vitro Cell Dev Biol, 28A:529-36, 1992). ATP leads to the induction of a variety of intracellular signaling pathways linked to mitogenic activity, including stimulation of immediate early genes such as c-fos and c-jun, binding of transcription activator protein-1 (AP-1) complex to DNA, and activation of extracellular signal-regulated protein kinase (Bolego, et al., Br J Pharmacol, 121:1692-9, 1997; Chen and Sun, Neurochem Res, 23:543-50, 1998; Priller, et al., Neuroscience, 85:521-5, 1998; Wu, et al., Mol Pharmacol, 53:346, 1998). In the nervous system, glial-specific activation of AP-1 complex, immediate early genes, and ras-MAPK pathway is associated with a primary response of glial cells to perturbation and trauma arising from environmental and genetic etiologies (Segal and Greenberg, Annu Rev Neurosci, 19:463-89, 1996). Activation of these intracellular signaling pathways in glial cells is thought to represent an underlying, adaptive response to neuronal stress and enables glia to mount a neuroprotective response to provide trophic conditioning to neurons from subsequent injury and damage (Segal and Greenberg, Annu Rev Neurosci, 19:463-89, 1996).
Glia often undergo phenotypic changes in response to stress and injury, including proliferation and hypertrophy in a process known a reactive gliosis, which involves upregulation of glial fibrillary acidic protein (GFAP) and elongation of gliotic processes (Eddleston and Mucke, Neuroscience, 54:15-36, 1993; Hatten, et al., Glia, 4:233-43, 1991; Ridet, et al., Trends Neurosci, 20:5707, 1997). Recent work has shown that GFAP-positive, reactive astrocytes are required to protect neurons from extensive cell death in a mechanical injury model of neuronal degeneration in the central nervous system (Bush, et al., Neuron, 23:297-308, 1999). ATP has been shown to increase the expression of GFAP in rat cerebral cortical astrocytes and lengthen astrocytic processes (Abbracchio, et al., Int J Dev Neurosci, 13:685-93, 1995). Similar effects are observed by growth factors such as basic fibroblast growth factor and ciliary neurotrophic factor, which have been shown to confer neuroprotection in a variety of animal models of neurodegeneration (Segal and Greenberg, Annu Rev Neurosci, 19:463-89, 1996).
The following references disclose the compositions of P2Y receptor agonists and/or treatment of eye diseases. U.S. Pat. No. 5,900,407 (Yerxa et al.) discloses a method for the stimulation of tear secretion in a subject in need of treatment. The method comprises administering to the ocular surfaces of the subject a purinergic receptor agonist such as uridine 5′-triphosphate, cytidine 5′-triphosphate, adenosine 5′-triphosphate, or their analogs and derivatives, in an amount effective to stimulate tear fluid secretion. U.S. Pat. No. 5,837,861 (Pendergast et al.) discloses P2Y2 purinergic receptors of dinucleotide polyphosphates having structure of Formula I, wherein X is oxygen, methylene, or difluoromethylene; n=0 or 1; m=0 or 1; n+m=0, 1 or 2; and B and B′ are each independently a purine residue or a pyrimidine residue linked through the 9- or 1-position. The compounds are useful in the treatment of chronic obstructive pulmonary diseases, bronchitis, certain pneumonias, cystic fibrosis, sinusitis, and otitis media. U.S. Pat. No. 5,763,447 is directed to a method of preventing or treating pneumonia, including ventilator-associated pneumonia, in a bedridden or immobilized subject in need of such treatment. The method comprises administering to the airways of the patient a purinergic receptor such as uridine 5′-triphosphate (UTP), P1, P4 -di(uridine-5′)tetraphosphate, or their analogs, in an amount effective to promote drainage of fluid in the congested airways. WO 99/09998 discloses a method of using uridine 5′-diphosphate and analogs thereof to treat lung disease. The compounds described in the above references ('407, '861, and '447 patents and WO 99/09998), which have purinergic receptor activity, are incorporated herein by reference. Boyer e al., (Br. J. Pharmacol. 118:1959 (1996)) synthesized and tested a series of chain-extended 2-thioether derivatives of adenosine monophosphate (AMP) as agonsists for activation of the phospholipase C-linked P2Y-receptor of turkey erythrocyte membranes, the adenylyl cyclase-linked P2Y-receptor of C6 rat glioma cells, and the cloned human P2U-receptor stably expressed in 1321N1 human astrocytoma cells.
There continues to be a need for methods useful in the treatment retinal degenerations. Such treatment would prevent or reduce the rate of retinal degeneration arising from multiple etiologies. Based on the cellular localization of P2Y receptors in the retina and the signaling pathways of P2Y receptors, we were motivated to explore the utility of P2Y receptor agonists in developing such a treatment.